Method of cross-linking peptides

ABSTRACT

A method of cross-linking a peptide to form a homopolymer of the peptide, to immobilize the peptide on a solid phase and to enhance antigenicity of the peptide is disclosed. The method comprises the steps of preparing a fusion peptide by incorporating a cross-linking segment including a tetrapeptide sequence QXK(S/T) into the peptide and cross-linking the peptide by a glutaminase.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to a method of cross-linkingpeptides through a novel amino acid sequence Q-X-K-(S/T), which wasinitially identified from mouse seminal vesicle secretion (SVS) IIIprotein. The peptides containing Q-X-K-(S/T) sequence may becross-linked by any transgluatminase (TGase).

[0003] 2. Description of the Related Art

[0004] Cross-linking of biomolecules has been noted for decadesthroughout the world of biotechnology. Cross-linking of proteins withdifferent functions can produce a new molecule with multi functions.Enzymes cross-link to a solid phase makes its activity retaining thatcan reduce manufactory costs. Biomolecules can be cross-linked bychemical reactions while these reactions are not very specific andprobably reduced the activity of enzymes. An alternative way tocross-link biomolecules is enzyme-catalyzed reaction.

[0005] Transgluatminase (TGase) is a kind of enzyme with the ability tocatalyze protein-protein cross-linking reaction. Transglutaminase(TGase) was first reported by Heinrich Waelsch in 1959. He isolated theenzyme from the liver of guinea pig. In the presence of calcium ion,this enzyme shows trans-amidation activity which binding glutamine inproteins to primary amines covalently. Therefore this enzyme wasdesigned as transglutaminase (EC.2.3.2.13). In recent years, moretransglutaminases have been purified from different spices and differenttissues. Until now, TGases can be divided into five types: (1) tissuetype TGase (TG2), which is commonly expressed in all kinds of tissues,might involve cell-programmed death; (2) epidermal TGase (TG1), found inthe wounded epidermal tissues, enable epidermal proteins to cross-linkinto keratin; (3) hair-follicle TGase, found in hair-follicle cell, cancross-link hair protein; (4) plasma factor XIII, when catalyzed bythrombin, it can cross-link fibrin to stabilize the structure ofthrombus; (5) prostate TGase (TG4), found in the coagulating gland ofrodent, can catalyze seminal vesicle secreted proteins to copulatoryplug. Although these different kinds of TGases have great difference insize and sequences, their catalytic mechanisms are similar. Each of themhas a cystine residue in their active site and the enzyme activity iscalcium dependent. TGase has great usages in industry. For examples:adding TGase to meat will increase its tenacity and savor in foodprocessing; Enzymes can be fixed by cross-linking of TGase in enzymeengineering; TGase has been used to construct tissues' frame in tissueengineering et cetera.

[0006] However, there are limits in industrial applications for TGase.First, most TGase was isolated from animal source and they are alsodifficult to prepare by recombinant techniques. Second, most TGases havespecificities to their substrates that limits the application of TGase.J. E. Folk and his group made a series of studies on tissue type TGaseof guinea pig's liver and human's plasma factor XIII (Gorman, J. J. andFolk, J. E. 1980. J. Biol. Chem. 2255,4419-427; Schhrode, J. and Folk,J. E. 1979. J. Biol. Chem. 254,653-661). Their works may help tounderstand the substrate specificity of TGase. TGase has two substrates:one is usually defined as glutamine in a peptides chain and serves as anacyl donor; the other should be a primary amine, which serves as an acylacceptor. TGases demand more specificity of acyl donor than of acylacceptor in usual. Therefore, lots of polyamines (spermine and histaminefor example) also can be acyl acceptors and covalently bind withproteins as a kind of post-translational modification.

[0007] Some works have been done to define the effective sites fromsubstrates of TGases to produce cross-linking peptides fragment. Forexample, in U.S. Pat. Nos. 5,428,014 and 5,939,385, peptide sequencesfrom human plasma fibrinogen have been studied. These peptide fragmentsare proved to be cross-linkable by human plasma factor XIII and thischaracteristic has been applied in tissue engineering. This inventiongeneralized an S1-Y-S2 formula from plasma fibrinogen. In this case, SIrepresents T-I-G-E-G-Q, Y is 0˜7 interval amino acids, and S2 isX-K-X-A-G-D-V (U.S. Pat. No. 5,428,014, claim 1). Yet, this inventiondidn't define characteristics and effects of the amino acids in Yposition. Moreover, the peptide fragments in this invention were onlyeffective under the action of human plasma factor XIII that limits theusage of other sources of TGases and also restricts the utilities ofthese peptide fragments. Besides, the length of the defmed fragment wastoo long and the reaction efficiency was low.

[0008] In the present invention, we have found a better substrate ofTGase from other sources. Seminal vesicles secretions of rodent havebeen reported as good substrate of TGase (Notides, A. C. andWilliams-Ashman, H. G. 1967. Proc. Natl. Acad. Sci. U.S.A. 58,1991-1995). Notides and Williams-Ashman found a protein (18 kDa)secreted from guinea pig's seminal vesicle. This protein can readily bepolymerized by a TGase secreted from coagulating gland. Following studyalso proved that SVS II protein from mouse and rat seminal vesiclesecretions are substrates of TGase (Harris, S. E. et. al. 1990. J. Biol.Chem. 265, 9896-9903; Lundwall, A. et al. 1997. Eur. J. Biochem.249,39-44). Though human seminal secretions will not solidify to becomecopulatory plugs, it has been proved that SgI and SgII proteins fromhuman seminal vesicle are also substrates of TGase (Peter, A. et. A1.1998. Eur. J. Biochem. 252, 216-221). However, the molecular mechanismof these proteins have never been studied. In this invention, weisolated a new protein, SVS III, from mouse seminal vesicle and provedit a good substrate of transglutaminase. The present invention alsoprovides an effective sequence from SVS III and related applications.

SUMMARY OF THE INVENTION

[0009] The purpose of this invention is to take the reactive site in SVSIII protein as the substrate of transglutaminase. It provides a novelway to cross-link, fix or polymerize proteins by TGase. The presentinvention defines the reactive site of TGase from mouse SVS III genesequence (SEQ ID NO: 1). The minimum effective unit of reaction siteincludes four amino acids (SEQ ID NO: 2), which are defined asQ-X-K-(S/T). Q represents glutamine, X represents aliphatic side chainof amino acids (Leu, Val, Ile, Ala etc., for example); K is lysine;(S/T) can be serine or threonine. The examples of this invention claimthat the minimum effective unit can be the substrate of TGase.

[0010] When peptides have repetitive minimum effective units (SEQ ID NO:3 including five minimum effective units, for example), they are bettersubstrates of TGase.

[0011] Examples in this invention claim that the rearranged sequence ofminimum effective unit (SEQ ID NO: 4) also can be the substrate ofTGase. “Cross-linking fragment” is defined as peptides with one or moreminimum effective unit or with deformed sequence. Accordingly, thepresent invention encompasses a peptide for cross-linking, comprising a-QX- sequence at the N-terminal and a -XK(S/T) sequence at theC-terminal, wherein Q is glutamine, X is an amino acid having analiphatic side chain, K is lysine and (S/T) is either serine orthreonine.

[0012] Examples in this invention also prove that the cross-linkingfragment is a good substrate for TGase from different sources includingplasma factor XIII, TGase of guinea pig and TGase in mouse coagulateland secretion.

[0013] This peptide fragment can be synthesized directly (Merrifiedld,R. B. 1963. J. Amer. Chem. Soc. 85, 2149-2154), or produced byconstructing the cDNA sequence in recombinant plasmid to produce fusionprotein. In examples of this invention, we construct the cDNA sequencein recombinant plasmid to produce fusion protein with this cross-linkingfragment. Therefore, the fusion protein has the ability to cross-link byTGase. In examples of this invention, we explain how to produce a fusionprotein with cross-linking fragment.

[0014] The fusion protein containing the cross-linking fragment can becross-linked to the plastic surface with primary amine by TGase, whichretains its enzyme activity.

[0015] In examples of this invention, we also proved that fusion proteinwith the cross-linking fragment could become polymer, which can inducestronger immune reaction when it is injected into animals as an antigen.This method makes an improvement on the production of vaccines.

[0016] Other objects and features of the present invention will becomeapparent from the following detailed description considered inconjunction with the accompanying drawings. It is to be understood,however, that the drawings are designed solely for purposes ofillustration and not as a definition of the limits of the invention, forwhich reference should be made to the appended claims. It should befurther understood that the drawings are not necessarily drawn to scaleand that, unless otherwise indicated, they are merely intended toconceptually illustrate the structures and procedures described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In the drawings:

[0018]FIG. 1 depicts construction of the recombinant plasmid, where (A)is a map of the recombinant expression vector, pGEX-4T and the sites ofrestriction enzymes recognized, and (B) shows sequences of each pair ofsynthesized nucleotides in each clone were aligned in the annealed form;

[0019]FIG. 2 shows cross-linking of GST fusion proteins by guinea pigliver transglutaminase, where GST protein (lanes 1 and 2), FP#1 (lanes 3and 4), FP#2 (lanes 5 and 6) and FP#3 (lanes 7 and 8) were cross-linkedby guinea pig liver transglutaminase in a reaction buffer (50 mMTris-HCl, 150 mM NaCl and 7.5 mM CaCl2) with (lanes 1, 3, 5, and 7) orwithout (lanes 2, 4, 6 and 8) 50 mM EDTA;

[0020]FIG. 3 shows that a fusion protein containing QXKS/T is a goodsubstrate for different sources of transglutaminase, where FP#1 wascross-linked by different sources of transglutaminases, including mousecoagulating gland fluid (C), human blood factor XIII (F), and guinea pigtransglutaminase (T), in a reaction buffer (50 mM Tris-HCl, 150 mM NaCland 7.5 mM CaCl2) with (lanes 1, 3, and 5) or without (lanes 2, 4, and6) 50 mM EDTA; and

[0021]FIG. 4 shows that GST fused with QXKS/T tandem repeats segment canbe fixed on the surface of primary amine containing microplate, where(A) shows GST activity in the reaction mixture after the cross-linkingreaction, and (B) shows GST activity in the well of microplate after thecross-linking reaction (see “example 2” for detail experimentcondition). This Data represent the means of three experiments, anderror bars represent S.D.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

[0022] As used herein, the terms “cross-linking fragment”,“cross-linking sequence”, “cross-linking segment”, or “minimum effectiveunit” means the peptide having a Q-X-K-(S/T) sequence, wherein Qrepresents glutamine, X represents amino acids having an aliphatic sidechain such as Leu, Val, Ile, Ala etc.; K is lysine; (S/T) can be eitherserine or threonine; the term “fusion protein”, “fusion peptide” or“fusion polypeptide” means a peptide containing fragments from differentorigins.

[0023] “TGase” represents transglutaminase;

[0024] “GST” represents glutathione S-transferase.

[0025] The present invention is further explained and illustrated in thefollowing examples, which represent particular embodiments of, but notlimitations to, the present invention.

EXAMPLE 1 Preparation of Fusion Proteins Containing Cross-linkingEffective Sequence

[0026] All fusion proteins identified in the present invention wereprepared by inserting a cross-linking fragment into GST sequence byrecombinant DNA technology. E. Coli expression vector, pGEX4-T(Amersham-Pharmacia, Freiburg, Germany), was used to produce fusionproteins. Restriction enzymes, Bam HI and EcoRI, were used to cut thepGEX4-T vector. To remove the restriction enzymes and small nucleotides,the vector was then purified by 1% agarose gel electrophoresis andrecovered from the gel by QiagelElution kit (Qiagen, Hilden, Germany). Asense and an anti-sense DNA fragment encoded each protein fragment weresynthesized based on mouse SVS III cDNA (SEQ ID NO: 1). At the beginningand the end of each DNA fragment, a BamHI site and an EcoRI site wereadded during nucleotide synthesizing (cf. FIG. 1). 20 μl of both strandsof the synthesized oligonucleotides, in the concentration of 100 μM,were mixed together and heated to 95° C. for 10 min, then annealed at55° C. for 10 min and room temperature for another 10 min. The annealedinsert DNA fragment was mix with the BamHI/EcoRI-treated pGEX4-T vectorand reacted by T4 DNA ligase at 4° C. overnight. The reaction mixturewas use to transform host cells, E. Coli strain JM109, by conventionaltransformation technology. Positive clones, which were able to produce afusion polypeptide were screened and identified by DNA sequencing.

[0027] To produce the recombinant proteins, each bacteria clone wastransferred into 100 ml of LB broth containing 100, μg/ml ampicillin andcultured at 37° C. with 200 rpm shaking overnight. The bacteria brothwas subcultured in 900 ml LB medium containing 100 μg/ml ampicillin inthe next morning. When OD₆₀₀ of the bacteria broth reached 0.6, IPTG(0.5 mM of final conc.) were added into the broth and continuouslycultured at 37° C. with 200 rpm shaking for 5 hours. Then the broth wascentrifuged at 5,000 rpm for 10 min and discarded the supernatant. Thepellet was resuspensed by 10 ml of phosphate buffer saline and mixedwith complete protease inhibitor cocktail (Roch, Germany). The bacteriasuspension was sonicated for 5 min and the cell lysate was centrifugedat 15,000 rpm for 20 min. The supernatant, so-called the crude extract,was collected for further purification.

[0028] The fusion proteins were purified from the crude extract byaffinity chromatography on a column of glutathione agarose bead(Amersham-Pharmacia, Freiburg, Germany). Glutathione agarose waspackaged into a column with 20 mm inner diagram. The height of the gelwas 40 mm. 50 ml of phosphate buffer saline was flowed through thecolumn for equivalence. The crude extract was loaded into the column andthen another 50 ml of PBS was flowed through to wash out unboundproteins. Finally, 20 ml of elution buffer (10 mM reduction formglutathione in PBS) was applied into the column and the purified proteinwas collected. After dialysis and measuring protein concentration, therecombinant proteins were adjusted to 1 μg/ml and stored in −20° C.

EXAMPLE 2 Cross-Linking Ability of The Fusion Proteins

[0029] To test the cross-linking abilities of different fusion proteinsproduced in Example 1, each fusion protein was mixed with tissuetransglutaminase from guinea pig's liver in 40 μl reaction buffer (50 mMTris-HCl, 150 mM NaCl and 7.5 mM CaCl₂ pH7.5) and the reaction mixturewas incubated at 37° C. for 1 hour. Since the action of transglutaminaseis calcium dependent, every experiment had a control, which substitute50 mM EDTA for 7.5 mM CaCl₂. After 1 hour of incubation, 40 μl of 2×Laemmli sample buffer was added to stop the reaction. Each reactionresult was resolved by SDS-PAGE.

[0030] As shown in FIG. 2, GST itself is not a substrate of TGase(lane 1) and was not cross-linked by the enzyme, while GST fused withinsert SEQ ID NO:2 (Fusion Protein No:1, FP#1, lane 3), SEQ ID NO:3(FP#2, lane 5) and SEQ ID NO:4 (FP#3, lane 7) showed the ability to becross-linked by transglutaminase. The presence of EDTA in the reactionmixture prevents the fusion proteins to form polymers, because of thelack of calcium ion. Note worthily, the enzyme-catalyzed cross-links ofFP#2 were very striking (cf. lane 5 of FIG. 2). There were almost noFP#2 monomer left after the enzyme reaction. Based on the relationbetween molecular size and protein mobility on SDS/PAGE, dimers,trimers, tetramers, pentamers and hexamers of FP#2 were clearlyidentified. Homopolymers larger than hexamers were also detected.Apparently, FP#2 was intermolecularly cross-linked by the enzymereaction. FP#1 was cross-linked to a dimer by the enzyme reaction (cf.lane 3 of FIG. 2), manifesting the transglutaminase substrate activityof the short peptide QIKS. Likewise, the enzyme was able to cross-linkFP#3, which was a mutant of FP#2 with a QI at the N-terminal and a KS atthe C-terminal of the cross-linking segment but the inner glutamine (Q)and lysine (K) residues were replaced by glycine residues. Apparently,the four-peptide segment of QXK(S/T) is the essential sequence forcross-linking by transglutaminase. While one segment of QXK(S/T) issufficient for the transglutaminase-catalyzed protein cross-linking, themore QXK(S/T) repeats the fusion protein contains, the stronger is thecross-linking ability. The sequence of QXK(S/T) can also be rearrangedand still maintain its cross-linking ability by transglutaminase.

EXAMPLE 3 Cross-linking Abilities of the Fusion Proteins byTransglutaminases from Different Sources

[0031] Fusion proteins containing QXK(S/T) sequences are not onlycross-linked by guinea pig liver transglutaminase but also goodsubstrates to other types of transglutaminases. Thrombin-activatedfactor XIII (F), guinea pig liver transglutaminase (T), or mousecoagulating gland fluid (M) was incubated with FP#1 (15 μg) in 40 μlreaction buffer (50 mM Tris-HCl, 150 mM NaCl and 7.5 mM CaCl₂ pH7.5) andincubated at 37° C. for 1 hour. Since the action of transglutaminase iscalcium dependent, every experiment had a control, which 7.5 mM CaCl₂was substituted by 50 mM EDTA. After 1 hour of incubation, 40 μl of 2×Laemmli sample buffer was added to stop the reaction and the reactionmixture was resolved by SDS/PAGE on a 14% gel slab.

[0032] As shown in FIG. 3, FP#1, with the essential sequence of QXKS/T,is a good substrate of transglutaminases from different sources,including mouse coagulating gland transglutaminase (TG4), human bloodfactor XIII and guinea pig liver transglutaminase (TG2).

EXAMPLE 4 Fixation of Fusion Protein Containing QXK(S/T) Sequence to aSolid Phase

[0033] Since transglutaminase has the ability to transfer an acyl groupfrom a molecule to a primary amine so as to form a covalent bond, it ispossible to fix a fusion protein which containing QXK(S/T) sequence to asolid phase having primary amine on its surface by the action oftransglutaminase. In a volume of 50 μl, the reaction mixture contained 1μg of FP#2, 0.1 μg of guinea pig liver transglutaminase, 50 mM Tris-HCl,150 mM NaCl and 7.5 mM CaCl₂ in pH 7.5. The reaction mixture was loadedinto wells of a microplate which contains primary amine on the surface(COSTAR amine surface stripwell, Corning, USA.) and incubated at 37° C.for 2 hrs. As a control, CaCl₂ in the reaction was replaced by 50 mMEDTA. After reaction was complete, the supernatant in each well wascollected to a microcentrifuge tube and the wells were washed twice byPBS. The supernatant was then mixed with 1 ml of assay reagent (100 mMpotassium phosphate buffer pH6.5, 1 mM glutathione, 1 mM1-Chloro-2,4-dinitrobezene) to test the enzyme activity of GST. Thereaction was carried out at 37° C. for 5 min and the absorption at 340nm was observed. The enzyme activity in the wells were also tested bypouring 100 μl of assay reagent (100 mM potassium phosphate bufferpH6.5, 1 mM glutathione, 1 mM 1-Chloro-2,4-dinitrobezene) into each welland incubated the plate at 37° C. for 5 min. The solution of each wellwas collected and its absorption at 340 nm was measured. A strongerabsorption represents a higher enzyme activity. The highest enzymeactivity was found in the control supernatant (FIG. 4A). However, underthe action of transglutaminase, the GST activity of FP#2 was remained inthe well of microplate (FIG. 4B). Thus, it demonstrates thattransglutaminase can be used to fix a fusion protein having QXK(S/T)sequence to a solid phase.

EXAMPLE 5 Polymerization of the Fusion Proteins to Improve Antigenicity

[0034] Polymerization of an antigen containing QXK(S/T) sequence bytransglutaminase can improve antigenicity. In a volume of 50 μl, thereaction mixture contained 50 μg of FP#2 as an antigen, 1 μg of guineapig liver transglutaminase, 50 mM Tris-HCl, 150 mM NaCl and 7.5 mM CaCl₂in pH 7.5. The reaction was carried on at 37° C. for 1 hrs. The CaCl₂ inthe reaction was replaced by 50 mM EDTA in the control. After thereaction was completed, an equal volume of Freund's incomplete adjuvantwas added into each reaction mixture and mixed well with the reactionmixture to form an antigen injection mixture. These antigen injectionmixtures were used to inject 12-week-old female mice subcutaneously.Three weeks after the first boost, mice were challenged with the sameantigen and received the second challenge after another 3 weeks. Theantiserum was collected two weeks after the final challenge. The FP#2protein was resolved by SDS-PAGE and transferred to a nitrocellulosemembrane. After transfer, the protein blots were immunodetected by theWestern blot procedure, using the antiserum as the primary antibodydiluted to 1:10000 in a blocking solution (5% nonfat skimmed milk inPBS), and a goat anti-mouse IgG was conjugated with horseradishperoxidase as the secondary antibody diluted to 1:10000 in the blockingsolution. The enzyme-staining bands were enhanced by chemiluminescencedetection using an ECL kit (Amersham-Pharmacia, Freiburg, Germany)according to the manufacturer's instruction. The result showed a strongimmunoreaction to the antigen (FP#2), while the signal in the controlserum was weak. Apparently, the antigenicity of the antigen was improvedby the polymerization of the FP#2 through the action oftransglutaminase.

[0035] Thus, while there have shown and described and pointed outfundamental novel features of the invention as applied to a preferredembodiment thereof, it will be understood that various omissions andsubstitutions and changes in the form and details of the devicesillustrated, and in their operation, may be made by those skilled in theart without departing from the spirit of the invention. For example, itis expressly intended that all combinations of those elements and/ormethod steps which perform substantially the same function insubstantially the same way to achieve the same results are within thescope of the invention. Moreover, it should be recognized thatstructures and/or elements and/or method steps shown and/or described inconnection with any disclosed form or embodiment of the invention may beincorporated in any other disclosed or described or suggested form orembodiment as a general matter of design choice. It is the intention,therefore, to be limited only as indicated by the scope of the claimsappended hereto.

1 15 1 265 PRT Murine sp. 1 Met Lys Ser Ile Phe Phe Ser Leu Ser Leu LeuLeu Leu Leu Glu Lys 1 5 10 15 Lys Ala Ala Gly Ile Glu Leu Tyr Ala GlyGly Thr Lys Gly His Phe 20 25 30 Leu Val Lys Thr Ser Pro Leu Met Phe IleGly Lys Asn Gln Phe Leu 35 40 45 Tyr Gly His Lys Glu Glu Gln Glu Glu AlaPro Glu Glu Ser Ile Phe 50 55 60 Val Gln Thr Lys His His Ala Tyr Gly GlnAsp Ala Asp Ala Asp Met 65 70 75 80 Gly Gly Ala Leu Ser Ser Gln Glu LeuThr Ser Leu Lys Glu Asp Ile 85 90 95 Val Cys Glu Glu Glu Asp Glu Leu AlaGln Gln Lys Ser Gln Leu Pro 100 105 110 Ser Gln Ser Gln Ile Lys Ser GlnThr Gln Val Lys Ser Tyr Ala Ala 115 120 125 Gln Leu Lys Ser Gln Pro GlyGln Leu Lys Thr Ile Gly Gln Val Lys 130 135 140 Ser Gln Thr Met Leu LysSer His Gly Ala Pro Leu Lys Ser Phe Lys 145 150 155 160 Ala Arg Leu AsnLeu Arg Glu Asp Ile Pro Gln Gln Val Lys Gly Arg 165 170 175 Gly Tyr GlyLeu Ala Glu Asp Leu Ala Gln Val Arg Gln Gln Pro Ala 180 185 190 Lys ValHis Arg Leu Lys Gly Lys His Arg Gln Ser Arg Lys Thr Ala 195 200 205 AlaPhe Tyr Pro Gln Phe Arg Arg His Ser Arg Pro Tyr Pro Arg Tyr 210 215 220Phe Val Gln Phe Gln Glu Gln Leu Gln Gly Ser Val His His Thr Lys 225 230235 240 Ser Phe Tyr Pro Gly Pro Gly Met Cys Tyr Cys Pro Arg Gly Gly Val245 250 255 Ile Leu Tyr Gln Asp Ala Phe Thr Asp 260 265 2 4 PRTArtificial Sequence Description of Artificial Sequence Synthetic peptide2 Gln Ile Lys Ser 1 3 30 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 3 Gln Ile Lys Ser Gln Thr Gln ValLys Ser Tyr Ala Ala Gln Leu Lys 1 5 10 15 Ser Gln Pro Gly Gln Leu LysThr Ile Gly Gln Val Lys Ser 20 25 30 4 30 PRT Artificial SequenceDescription of Artificial Sequence Synthetic peptide 4 Gln Ile Gly SerGly Thr Gly Val Gly Ser Tyr Ala Ala Gly Leu Gly 1 5 10 15 Ser Gly ProGly Gly Leu Gly Thr Ile Gly Gly Val Lys Ser 20 25 30 5 18 DNA ArtificialSequence Description of Artificial Sequence Primer 5 gatcccaaat aaaatccg18 6 96 DNA Artificial Sequence Description of Artificial SequenceSynthetic nucleotide sequence 6 gatcccaaat aaaatcccaa actcaagtaaaatcctacgc agcccaactg aagtcccaac 60 caggccagct aaaaaccata gggcaggtgaagtcag 96 7 96 DNA Artificial Sequence Description of ArtificialSequence Synthetic nucleotide sequence 7 gatcccaaat aggttccggcactggggtag gttcctacgc agccggcctg ggttccgggc 60 caggcggtct aggtaccatagggggcgtga agtcag 96 8 57 DNA Artificial Sequence CDS (1)..(54)Description of Artificial Sequence Primer 8 ctg gtt ccg cgt gga tcc ccagga att ccc ggg tcg act cga gcg gcc 48 Leu Val Pro Arg Gly Ser Pro GlyIle Pro Gly Ser Thr Arg Ala Ala 1 5 10 15 gca tcg tga 57 Ala Ser 9 18PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 9 Leu Val Pro Arg Gly Ser Pro Gly Ile Pro Gly Ser Thr Arg AlaAla 1 5 10 15 Ala Ser 10 6 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 10 Thr Ile Gly Glu Gly Gln 1 5 117 PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 11 Xaa Lys Xaa Ala Gly Asp Val 1 5 12 18 DNA Artificial SequenceDescription of Artificial Sequence Primer 12 aattcggatt ttatttgg 18 1396 DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence 13 aattctgact tcacctgccc tatggttttt agctggcctggttgggactt cagttgggct 60 gcgtaggatt ttacttgagt ttgggatttt atttgg 96 1495 DNA Artificial Sequence Description of Artificial Sequence Syntheticnucleotide sequence 14 aattctgact tcacgccccc tatggtacct agacgcctggcccggaaccc aggccggctg 60 cgtaggaacc taccccagtg ccggaaccta tttgg 95 15 4PRT Artificial Sequence Description of Artificial Sequence Syntheticpeptide 15 Gln Xaa Lys Xaa 1

We claim:
 1. A method of cross-linking a peptide to form polymers ofsaid peptide, comprising the steps of (a) incorporating a cross-linkingsegment containing a Q-X-K-(S/T) sequence into said peptide to form afusion polypeptide, wherein Q is glutamine, X is an amino acid having analiphatic side chain, K is lysine and (S/T) is either serine orthreonine; and (b) cross-linking said fusion polypeptide by atransglutaminase in a cross-linking reaction buffer to form saidpolymers.
 2. The method of claim 1, wherein said cross-linking segmentcomprises at least two tandem repeats of said Q-X-K-(S/T) sequence. 3.The method of claim 2, wherein said cross-linking segment comprises SEQID No:
 3. 4. The method of claim 1, wherein said Q-X-K-(S/T) sequence isSEQ ID No:
 2. 5. The method of claim 1, wherein said transglutaminase isfrom mouse coagulating gland fluid.
 6. The method of claim 1, whereinsaid transglutaminase is from human blood factor XIII.
 7. The method ofclaim 1, wherein said transglutaminase is guinea pig livertransglutaminase.
 8. A method of cross-linking a peptide to formpolymers of said peptide, comprising the steps of incorporating across-linking segment containing SEQ ID No: 4 into said peptide to forma fusion polypeptide and cross-linking said fusion polypeptide by atransglutaminase in a cross-linking reaction buffer to form saidpolymers.
 9. A method of immobilize a peptide on a solid phase having asurface containing a primary amine moiety, comprising the steps of (a)preparing a fusion polypeptide by incorporating a cross-linking segmentcontaining a Q-X-K-(S/T) sequence into said peptide, wherein Q isglutamine, X is an amino acid having an aliphatic side chain, K islysine and (S/T) is either serine or threonine; and (b) cross-linkingsaid fusion polypeptide with the surface of said solid phase in thepresence of a cross-linking buffer by a transglutaminase to immobilizesaid fusion polypeptide.
 10. The method of claim 9, wherein saidcross-linking segment comprises at least two tandem repeats of saidQ-X-K-(S/T) sequence.
 11. The method of claim 10, wherein saidcross-linking segment comprises SEQ ID No:
 3. 12. The method of claim 9,wherein said Q-X-K-(S/T) sequence is SEQ ID No:
 2. 13. The method ofclaim 9, wherein said transglutaminase is from mouse coagulating glandfluid.
 14. The method of claim 9, wherein said transglutaminase is fromhuman blood factor XIII.
 15. The method of claim 9, wherein saidtransglutaminase is guinea pig liver transglutaminase.
 16. A method ofenhancing antigenicity of a peptide, comprising the steps of (a)preparing a fusion polypeptide by incorporating a cross-linking segmentcontaining a Q-X-K-(S/T) sequence into said peptide, wherein Q isglutamine, X is an amino acid having an aliphatic side chain, K islysine and (S/T) is either serine or threonine; (b) cross-linking saidfusion polypeptide by a transglutaminase in a cross-linking buffer toform polymers of said peptide; and (c) immunizing an animal by injectingsaid animal with said polymers of said peptide to produce an antibodyagainst said peptide.
 17. The method of claim 16, wherein saidcross-linking segment comprises at least two tandem repeats of saidQ-X-K-(S/T) sequence.
 18. The method of claim 17, wherein saidcross-linking segment comprises SEQ ID No:
 3. 19. The method of claim16, wherein said Q-X-K-(S/T) sequence is SEQ ID No:
 2. 20. The method ofclaim 16, wherein said transglutaminase is from mouse coagulating glandfluid.
 21. The method of claim 16, wherein said transglutaminase is fromhuman blood factor XIII.
 22. The method of claim 16, wherein saidtransglutaminase is guinea pig liver transglutaminase.
 23. A peptide forcross-linking, comprising a -QX- sequence and a -XK(S/T) sequence,wherein Q is glutamine, X is an amino acid having an aliphatic sidechain, K is lysine and (S/T) is either serine or threonine, said -QK-sequence and said -XK(S/T) sequence being spaced apart by at most 25amino acid residues.
 24. The peptide of claim 23 having a sequenceidentical to SEQ ID No:
 4. 25. A fusion polypeptide, comprising across-linking segment containing a Q-X-K-(S/T) sequence, wherein Q isglutamine, X is an amino acid having an aliphatic side chain, K islysine and (S/T) is either serine or threonine.
 26. The fusionpolypeptide of claim 25, wherein said cross-linking segment comprises atleast two tandem repeats of said Q-X-K-(S/T) sequence.
 27. The fusionpolypeptide of claim 26, wherein said cross-linking segment comprisesSEQ ID No:
 3. 28. The fusion polypeptide of claim 25, wherein saidQ-X-K-(S/T) sequence is SEQ ID No:
 2. 29. A fusion polypeptide,comprising a cross-linking segment containing a -QX- sequence and a-XK(S/T)- sequence, wherein Q is glutamine, X is an amino acid having analiphatic side chain, K is lysine and (S/T) is either serine orthreonine, said -QK- sequence and said -XK(S/T) sequence being spacedapart by at most 25 amino acid residues
 30. The fusion peptide of claim23, wherein said cross-linking segment has a sequence identical to SEQID No: 4.